As one of important fundamental means used in the field of genetic engineering, there may be mentioned detection (detection includes identification and quantification) of a nucleic acid (a nucleic acid includes a nucleic acid and a peptide nucleic acid) having a specific base sequence present in a sample mixture. By detecting the presence of a nucleic acid or a gene having a specific base sequence, it becomes possible to elucidate expression of a gene of interest and develop novel drugs, or to select cells or individuals transfected with a desired gene in gene recombination. It also becomes possible to diagnose or predict a disease before the onset or during the early phase of the disease by conducting genetic diagnosis and detecting any human genetic defect or alteration causing the disease.
In this way, detection of a nucleic acid having a specific base sequence present in a sample mixture is a fundamental means which is widely used in the field of genetic engineering, and for example, the following methods are known.
Initially, nucleic acid is extracted from cells that are desired for examination and a sample mixture solution of the nucleic acid is obtained. If needed, the nucleic acid is cleaved with appropriate restriction enzymes, and then subjected to electrophoresis, and membrane blotting is performed using the gel obtained therefrom. Subsequently, for a nucleic acid having a specific base sequence which is the aim of detection, a nucleic acid probe having a base sequence that is complementary to the specific base sequence is provided. Then, this nucleic acid probe is hybridized with the blotted nucleic acid. This nucleic acid probe is labeled in advance so as to become detectable. For example, the nucleic acid probe is labeled with a radioisotope. Thereby, a band of the nucleic acid hybridized with the nucleic acid probe is detected by autoradiography to confirm the presence of the target nucleic acid having a specific base sequence. This method, which is referred to as Northern Blotting in terms of RNA, and as Southern Blotting in terms of DNA, is still increasingly used while undergoing various modifications (description on the method is found in, for example, “Molecular Biology of the Cell” 4th Ed., translated and supervised by Keiko Nakamura and Kenichi Matsubara, Newton Press, 2004, 494-500).
Furthermore, particularly in the recent technologies of genetic diagnosis, DNA chips (DNA microarrays) are used in the detection of nucleic acids having specific base sequences. A DNA chip has DNA probes having various base sequences immobilized and arranged in arrays in each of many compartments on a substrate. A mixture of DNA derived from a test subject is provided by labeling the DNA in advance, for example, by labeling the DNA with a fluorescent dye. This test subject-derived DNA mixture is added dropwise to the DNA chip to induce hybridization. In the test subject-derived DNA mixture, DNAs containing base sequences that are complementary to the DNA probes become fixed on the substrate of the DNA chip through the hybridization. These fixed test subject-derived DNAs are detected, for example, by means of the previous labeling with a fluorescent dye. In this method, various modifications are being achieved, such as conditioning of DNA, use of RNA, or normalization through competitive implementation of hybridization (description on the method is found in, for example, “Molecular Biology of the Cell” 4th Ed., translated and supervised by Keiko Nakamura and Kenichi Matsubara, Newton Press, 2004, 533-535).
In this way, from the conventional methods as well as to the recent technologies, detection of a nucleic acid having a specific base sequence has long utilized hybridization with a nucleic acid probe having a complementary base sequence as the fundamental principle for enabling specific recognition of complementary base sequences. However, under the actual conditions for hybridization, it is difficult for the binding between a nucleic acid and a nucleic acid probe molecule to occur only with the nucleic acid having the target base sequence to hybridize a perfectly complementary strand. That is, it is known that formation of imperfect hybrid including certain mismatches occurs even with a nucleic acid having a non-target base sequence which is imperfect as a complementary strand, thus resulting in binding with a nucleic acid probe molecule. Such unintended binding with a nucleic acid probe emerges as noise in the detection stage. In order to increase the specificity of detection without the emergence of such noise, it is necessary to eliminate incomplete hybridization.
However, discrimination through hybridization makes use of the differences in thermal stability, and the difference between complete hybridization and incomplete hybridization lies only in the difference in thermal stability. For this reason, appropriate conditions for distinguishing the two states vary with the aimed base sequence, and even under appropriate conditions, the conditions for altering thermal stability have equal effects on both states. In other words, as long as the difference in the thermal stability of hybridization is used as the only principle for distinguishing the two states, certain noise has to be tolerated, while making a compromise in the balance between specificity and sensitivity of the detection.
Moreover, recently there is a demand for detection of a nucleic acid having a base sequence having a single base substitution for the purpose of development of novel drugs or genetic diagnosis. In particular, great expectation is posed on the technology of typing a single base polymorphism of DNA in the field of medical diagnostics. For this reason, it is especially demanded to achieve a balance between specificity to the extent of detecting a single base substitution, and a practicable sensitivity (S/N ratio).
Non-Patent Document 1: “Molecular Biology of the Cell” 4th Ed., translated and supervised by Keiko Nakamura and Kenichi Matsubara, Newton Press, 2004, 494-500
Non-Patent Document 2: “Molecular Biology of the Cell” 4th Ed., translated and supervised by Keiko Nakamura and Kenichi Matsubara, Newton Press, 2004, 533-535